This invention relates to the field of antibody detection, particularly rapid methods, devices and kits for detection and semi-quantitation of anti-adenovirus antibody.
The ability to detect antibodies to viruses such as adenovirus in a patient sample is increasingly becoming a prerequisite for optimal utilization of many modern health care technologies. This is especially relevant in two broad areas of health care: detection of infections based on presence of pathogen-specific antibodies, and xe2x80x9cpreppingxe2x80x9d of patients for therapeutic agent administration.
The pathogenesis of the ubiquitous adenovirus is well-documented. Many relatively minor illnesses are associated with adenovirus infections, including acute febrile pharyngitis, pharyngoconjunctival fever, acute respiratory disease, pneumonia, epidemic keratoconjunctivitis, pertussis-like syndrome, acute hemorrhagic cystitis, gastroenteritis, hepatitis and persistence of virus in urinary tract. Fields, et al., Fields"" VIROLOGY, Vol. 2, p. 2155 (3rd Ed.). A vaccine for the most severe of these diseases has been developed using a wild-type unattenuated replicating polyvalent vaccine comprised of adenovirus types 4 and 7.
Detection of anti-adenovirus antibodies is an important aspect of optimizing the administration, and hence therapeutic effects, of new therapeutic tools. This phenomenon results from two factors. First, many of the new therapeutic tools involve the use of adenoviral vector constructs. Second, it is now clear that the full potential of the virus-based therapeutic tools, specifically those that exploit the gene delivery advantages of adenovirus vector constructs, is hampered by the recipient""s immune response.
Adenoviruses also form the basis of some of the most innovative and potentially powerful disease-fighting tools. One such tool is gene therapy, in which a defective gene or sequence is supplanted with an exogenous sequence. This approach holds great potential in treating not only cancer, but many other diseases as well, including cystic fibrosis, anemia, hemophilia, diabetes, Hungtington""s disease, AIDS, abnormally high serum cholesterol levels, certain immune deficiencies, and many forms of cancer.
Gene therapy generally requires a delivery vehicle for the exogenous sequence, such as a viral vector. Recombinant adenovirus is one of the newly-developed viral agents that may be effective vectors against these diseases. For reviews, see Kim et al. (1996) Mol. Med. Today 12:519-527 and Smith et al. (1996) Gene Therapy 3:496-502.
In addition, in the cancer context, specific attenuated replication-competent viral vectors have been developed, in which selective replication in cancer cells preferentially destroys those cells. Various cell-specific replication-competent adenovirus constructs, which preferentially replicate (and thus destroy) certain cell types, are described in, for example, WO 95/19434, WO 98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 98/39464, WO 00/15820. Another attenuated replication-competent adenovirus is Onyx-015 adenovirus. Onyx-015 has a deletion in the E1B-55kDa protein, which normally inhibits the cellular p53 tumor suppressor protein. Onyx-015 can replicate in p53-deficient human cells, but does not replicate efficiently in p53-positive cells. Bischoff et al. (1996) Science 274:373-376; Heise et al. (1997) Nat. Med. 3:639-645.
The favorable factors that make adenoviruses a safe therapeutic agent include: (a) infection with adenovirus has minor clinical disease manifestations; (b) adenovirus has a stable well-described and characterized genome; (c) adenovirus is unable to integrate its viral DNA into host DNA; (d) adenovirus allows transient gene expression; (e) adenovirus is able to infect both dividing and non-dividing cells; (f) adenovirus can infect a variety of human cell types; (g) adenovirus is physically stable; and (h) adenovirus is amenable to high titer production.
There are 47 different serotypes of adenovirus, which are distinguishable by antibody reactivity to epitopes on the virion surface. Each serotype is assigned to one of five Subgroups (A-E). Members of a Subgroup can exchange genetic material (recombine) efficiently, but they do not recombine with members of a different Subgroup. Adenovirus types 1, 2, 5, and 6 are members of Subgroup C. Adenovirus type 5 (the type typically used in gene therapy and for other therapies) is associated with a self-limiting, febrile respiratory illness and ocular disease in humans. In long-term immunosuppressed individuals, adenovirus-5 is also associated with renal impairment, hepatic necrosis, and gastric erosions. Shields et al. (1985) New England J. Med. 312:529-533; Zahradnik et al. (1980) Am. J. Med. 68: 725-732. Adenovirus-5 and the other Subgroup C viruses have little or no oncogenic potential in mammals. Horowitz (1990) in Virology, (Raven Press, New York, 2nd Ed.) pp. 1679-1721.
While use of viral therapeutic agents such as adenovirus holds promise, there are a number of potentially significant barriers to their effectiveness. Two of the major limitations of virus-based vectors as therapeutic vehicles result from the subject""s immune response to the presence of the viral agents, namely (a) the inactivation of virus by pre-existing circulating antibodies to the virus, and (b) the reduced efficacy of repeat dosage by primary or secondary induction of humoral immunity. For example, a recent serological survey indicates that 57% of the adult population in the U.S. has neutralizing antibodies to adenovirus-5 with titers ranging from 1:2 to 1:512. Schulick et al. (1997) J. Clin. Invest. 99:209-219. Neutralizing antibodies are generated to specific antigenic determinants within 7-14 days following intravenous adenovirus injection. Zinkernagel (1996) Science 271:173-178. These antibodies are typically specific for proteins on the virion, such as capsid proteins and various glycoproteins. George-Fries et al. (1984) Virology 134(1):64-71; Fisher et al. (1997) Nat. Med. 3(3): 306-12; Eing et al. (1989) J. Med. Virol. 27(1):59-65; Highlander et al. (1987) J. Virol. 61(11)3356-64; Durali et al. (1998) J. Virol. 72(5):3547-53. Activation of CD4+ lymphocytes by adenovirus capsid proteins also leads to the up-regulation of MHC class 1 molecules in infected cells contributing to the production of neutralizing antibodies as well as the clearing of adenovirus infected cells by CTLs. Yang et al. (1995) J. Virol. 69:2004-2015. For many patients, a therapeutic adenovirus will elicit an amnestic humoral response and a CTL response further decreasing the efficacy of repeat intravenous treatment with the same virus. Since the majority of the human population has been exposed to adenovirus during their lifetime, pre-existing immunity could be a major obstacle to the use of adenoviral vectors. Such high prevalence of neutralizing antibodies to adenovirus in adult humans could inhibit adenovirus dissemination (to distant tumor sites for example) and greatly limit the effectiveness of adenovirus-based therapy in vivo. More difficult to quantify is the potential non-neutralizing antibodies to inactivate adenovirus by opsonization.
The effect of antibodies on viral dissemination is a major issue in determining the success of viral therapy using parenteral administration, especially since intravenous administration may be desirable for treatment for metastatic disease. Although recent studies have indicated that pre-existing antibody may not reduce the efficiency of intratumoral viral administration (in terms of tumor regression), virus dissemination appears to be greatly impeded by pre-existing circulating antibodies. One group found that transgene expression in the liver of adenovirus-immune animals following intratumoral injection was reduced more than 1000-fold compared to the transgene expression found in nxc3xa4ive mice. Bramson et al. (1997) Gene Therapy 4:1069-1-76. In another example, in mice 90% of viral vectors is eliminated within 24 hours of intravenous injection. Worgall et al. (1997) Hum. Gene Ther. 8:37-44. This finding was confirmed by quantitative analysis of viral DNA in liver, spleen and lung using Southern analysis over the first 70 hours post injection demonstrating a 90% elimination of vector. Christ et al. (1997) Immunology Letters 57:19-25. Schulick et al. (1997) found that if rats are immunized by prior intravenous exposure to adenovirus, a second intravenous injection of an adenovirus vector gave no evidence of recombinant gene expression three days after the attempted gene transfer. This observation could be reproduced even in the presence of low (i.e., 1:2) titers of neutralizing antibody to adenovirus. Schulick et al. (1997). Another group examined immune responses to an adenoviral vector and to the recombinant gene expression (xcex2-galactosidase protein) in four patients with lung cancer. Gahery-Segard et al. (1997) J. Clin. Invest. 100:2218-2226. In patient 1, a high level of neutralizing antibodies to adenovirus was detected before adenovirus-xcex2-gal injection (100% neutralization at 1:400), whereas it was low in patient 3 (30% neutralization at 1:400) and undetectable in the other two patients. Virus DNA was detected by PCR in tumor biopsies on day 30 and day 60 from all patients except patient 1. Thus, recent studies have established that pre-existing humoral immunity poses a significant barrier to viral half-life, dissemination and effectiveness, and pre-existing anti-adenovirus neutralizing antibodies limit the therapeutic efficacy of adenovirus-mediated therapy through intravenous administration.
To address the problem of pre-existing immunity, various strategies have been developed. One strategy has been to down-regulate pre-existing anti-adenovirus immunity by oral tolerization based on administration of adenoviral proteins. Ilan et al. (1997) J Clin. Invest. 99(5):1098-1106. A potentially more effective and efficacious strategy is apheresis, which is a process by which certain blood components are removed extracorporeally and the blood is reintroduced into an individual, as described in PCT application WO 00/20041.
Apheresis can enhance performance of therapeutic viral vectors by partially pre-empting the body""s immune defenses. This strategy, in turn, requires monitoring of humoral immunity before, during and after apheresis treatment. Towards that end, a rapid and convenient method for detection and/or semi-quantitation of anti-adenovirus antibody is particularly useful.
Numerous methods for detection and/or semi-quantitation of antibodies, including virus antibodies, are known. In the area of HIV (human immunodeficiency virus) antibody detection, published methods include a rapid enzyme immunoassay called Single Use Diagnostic System (SUDS) (see, e.g., Kassler, et al. (1995) J. Clin. Microbiol. 33:2899-2902; http://www.int-murex.com/virtop.htm; serological assays using non-denatured HIV antigenic determinants (U.S. Pat. No. 5,587,285); comb spotting detection using artificial synthetic polypeptide antigen (Chinese Patent Application No. 1195772); an assay based on pre-formation of a complex of antibody and enzyme-conjugated antibody binding protein (Austrian Patent Application No. 9400870); assay using polymer that has antigen fixed covalently to its surface through acid anhydride group (Japanese Patent Application No. A7012819); solid-phase assay for simultaneous detection of different antibodies (European Patent Application No. 402757); solid-phase immunoassay for simultaneous antigen and antibody detection (East German Patent Publication No. 272134); and an assay utilizing a non-human immune antibody reactive with an anti-human antibody as a positive control (PCT application WO 89/10980).
Various methods for detection and/or semi-quantitation of anti-adenovirus antibodies specifically are also known. One group has disclosed an enzyme-linked immunosorbent assay for the visual detection of adenoviral antibodies. Bode, et al. (1984) J. Virol. Methods, 8:111-121. The Bode method is based on immobilization of crude adenovirus preparations on nitrocellulose discs in microtitre plates. However, the method requires repetitive reagent additions to and removals from the reaction location, and the assay takes at least 4 hours to complete. Other methods include latex agglutination (e.g., Lengyel, A. et al. (1992) Acta Microbiol. Hung., 39(3-4):303-8); simultaneous detection of more than one antibody by formation of complexes with labeled binding partner followed by capture on solid phase (WO 97/01758); and counterimmunoelectrophoresis (e.g., Petric, M. et al. (1979) J. Clin. Microbiol., 10(2):256-8).
Other solid-phase immunoassays include general methods and apparatuses comprising solid support and absorbent material as described in U.S. Pat. Nos. 4,632,901 and 4,727,019. Neither of these publications discloses detection of anti-adenovirus antibodies with antigen adsorbed on solid support. U.S. Pat. No. 4,366,241 discloses concentrating zone method and apparatus for performing immunoassays. U.S. Pat. No. 5,486,452 discloses devices and kits for solid-phase immunoassays comprising a solid porous support where antigens or immunoglobulins are bound by direct application in a suitable geometry. U.S. Pat. No. 5,395,754 describes a membrane-based immunoassay method that includes having at least two sterically separate antigenic sites including at least one calibration zone.
It is clear from the foregoing discussion that rapid detection of anti-adenovirus antibodies in a subject sample is important and needed in monitoring, maximizing and ensuring efficacy of adenovirus-based therapy. The current invention provides rapid and convenient methods for detection and semi-quantitation of anti-adenovirus antibodies.
All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.
The present invention provides methods, devices and kits for detecting and/or semi-quantitating anti-adenovirus antibodies.
Accordingly, in one aspect, the invention provides a method for detecting anti-adenovirus antibody in a sample, comprising (a) contacting the sample (generally and usually a biological sample) with an antigen that specifically binds to anti-adenovirus antibody under conditions that permit formation of a stable antibody antigen complex, wherein the antigen is adsorbed on a membrane, and wherein the membrane is contacted with an absorbent; and (b) detecting any complex formed.
In one embodiment, the detection method comprises these steps: placing a wash buffer on the membrane; contacting a test sample with the pre-adsorbed antigen (i.e., antigen adsorbed on a membrane) to form a mixture of antigen and test sample; and sequentially placing a wash buffer, a detection molecule, and a wash buffer on the membrane.
Presence of anti-adenovirus antibody in the test sample is indicated by a detectable signal. In one embodiment, the membrane is a nitrocellulose paper-backed membrane. The absorbent is composed of material capable of absorbing, either by capillary action or otherwise, molecules that pass through the membrane. Preferably, the absorbent material is cellulose based. The test sample is any liquid that potentially contains anti-adenovirus antibody, the presence of which is to be determined with the invention. Preferably, the sample is plasma, serum or whole blood, for which detection of the presence of anti-adenovirus antibody is desired. The antigen is any substance that has antigenicity for anti-adenovirus antibodies. Adenoviruses are preferred. Even more preferred is whole/intact adenovirus, most preferably whole/intact adenovirus-5 (Ad5). Also preferred are adenovirus surface proteins. The detection molecule is any molecule that generates a detectable signal in the presence of anti-adenovirus antibodies. A preferred detection molecule is gold-conjugated Protein A comprising colloidal gold particles coated with protein A, BSA and PEG. Also preferred is to have the detection molecule in a buffer containing buffer, salts, protein, surfactant and preservative. Alternatively, the detection molecule is linked to an enzyme that is capable of catalyzing a chemical reaction that produces a detectable signal.
In another aspect, the invention provides a method for semi-quantitating anti-adenovirus antibody in a sample, comprising (a) contacting the sample with an antigen that specifically binds to anti-adenovirus antibody under conditions that permit formation of a stable antibody antigen complex, wherein the antigen is adsorbed at a membrane, and wherein the membrane is contacted with an absorbent; (b) detecting any complex formed; and (c) comparing a detectable signal, if any, of the antigen spot to a detectable signal of a reference sample spot, whereby anti-adenovirus antibody is semi-quantitated.
In one embodiment, the semi-quantitation method comprises these steps: placing a wash buffer on the membrane, contacting a test sample with the pre-adsorbed antigen; and sequentially placing a wash buffer, a detection molecule and a wash buffer on the membrane. Semiquantitation is achieved by comparing the signal intensities of antigen and reference sample spots. The reference sample is a substance that gives a detection signal equal or approximately equal to that of a known amount of anti-adenovirus antibody. Preferably, it is an antibody of the same Ig class or subclass as the anti-adenovirus antibody being semiquantitated. Human IgG is preferred. Other Ig""s or subclasses are also preferred, including, but not limited to, IgE, IgA, IgD and IgM. The wash buffer, membrane and antigen are as described above for the detection method.
In another aspect, the invention provides devices for detection or semi-quantitation of anti-adenovirus antibody in a sample, comprising a membrane and an absorbent, wherein the membrane and absorbent are contacted with each other and wherein at least one antigen that specifically binds anti-adenovirus antibody is adsorbed on the membrane.
In another aspect, the invention provides kits for detection or semi-quantitation of anti-adenovirus antibody in a sample, comprising a membrane, an absorbent, and an antigen that specifically binds to anti-adenovirus antibody, said kit in suitable packaging. In one embodiment, the membrane and absorbent are contacted with each other. In another embodiment, at least one antigen is adsorbed on the membrane. In another embodiment, at least one antigen is provided separately. In yet another embodiment, at least one reference sample is adsorbed on the membrane. In still another embodiment, at least one reference sample is provided separately.